Cancer-Cell Imaging Using Copper-Doped Zeolite Imidazole Framework-8 Nanocrystals Exhibiting Oxidative Catalytic Activity

Article abstract of DOI:10.1002/asia.201800749

Copper-doped zeolite imidazole framework-8 (Cu/ZIF-8) was prepared and its peroxidase-like oxidative catalytic activity was examined with a demonstration of its applicability for cancer-cell imaging. Through simple solution chemistry at room temperature, Cu/ZIF-8 nanocrystals were produced that catalytically oxidized an organic substrate of o-phenylenediamine in the presence of H₂O₂. In a similar manner to peroxidase, the Cu/ZIF-8 nanocrystals oxidized the substrate through a ping-pong mechanism with an activation energy of 59.2 kJ mol^?1. The doped Cu atoms functioned as active sites in which the active Cu intermediates were expected to be generated during the catalysis, whereas the undoped ZIF-8 did not show any oxidative activity. Cu/ZIF-8 nanocrystals exhibited low cell toxicity and displayed catalytic activity through interaction with H₂O₂ among various reactive oxygen species in a cancer cell. This oxidative activity in vitro allowed cancer-cell imaging by exploiting the photoluminescence emitted from the oxidized product of o-phenylenediamine, which was insignificant in the absence of the Cu/ZIF-8 nanocrystals. The results of this study suggest that the Cu/ZIF-8 nanocrystal is a promising catalyst for the analysis of the microbiological systems.

Full text of DOI:10.1002/asia.201800749

CHEMISTRY
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Accepted Article
Title: Cancer-Cell Imaging Using Copper-Doped Zeolite Imidazole
Framework-8 Nanocrystals Exhibiting Oxidative Catalytic Activity
Authors: Changjoon Keum, Sangwoo Park, and Sang-Yup Lee
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Cancer-Cell Imaging Using Copper-Doped Zeolite Imidazole
Framework-8 Nanocrystals Exhibiting Oxidative Catalytic Activity
Changjoon Keum,†, [a] Sangwoo Park,† ,[b] and Sang-Yup Lee*,[a]
Abstract: Copper-doped zeolite imidazole framework-8 (Cu/ZIF-8) is
prepared and its peroxidase-like oxidative catalytic activity is
examined with a demonstration of its applicability for cancer cell
imaging. Through simple solution chemistry at room temperature,
Cu/ZIF-8 nanocrystals are produced that catalytically oxidize an
organic substrate of o-phenylenediamine in the presence of H2O2. In
a similar manner to peroxidase, the Cu/ZIF-8 nanocrystals oxidize
the substrate through a ping-pong mechanism with an activation
energy of 59.2 kJ·mol-1. The doped Cu atoms work as active sites in
which the active Cu intermediates are expected to be generated
during the catalysis, whereas the undoped ZIF-8 does not show any
oxidative activity. Cu/ZIF-8 nanocrystals exhibit low cell toxicity and
display catalytic activity through interaction with H2O2 among various
reactive oxygen species in a cancer cell. This oxidative activity in
vitro allowed cancer-cell imaging exploiting the photoluminescence
emitted from the oxidized product of o-phenylenediamine, which was
insignificant in the absence of the Cu/ZIF-8 nanocrystals. The results
of this study imply that the Cu/ZIF-8 nanocrystal is a promising
catalyst for the analysis of the microbiological systems.
sonochemical[12] approaches, where toxic organic solvents and
other chemical additives were required with heat treatment
(~200 °C).[13–15]
In addition to its mild synthesis conditions, ZIF-8 is
advantageous for catalytic material preparation because of its
high porosity and multiple sites to create active centres in the
crystalline structure. By involving heterogeneous ionic or
molecular species in ZIF-8, catalytic activity can be displayed
from Zn ions through the synergetic effect of organic linker
molecules. Although only a small number of studies have been
reported, one simple way to afford catalytic activity is doping
heterogeneous metal ions through which Zn ions are substituted
with heterogeneous metal ions. In 2009, Schubert et al.
prepared cobalt- or copper-doped bimetallic ZIF-1 (Zn(im2)2, im:
imidazole) by acidic hydrothermal synthesis at 80–150 °C.
Doping of these transition-metal ions to ZIF-1 resulted in the
alteration of the crystalline structure and the expression of the
paramagnetic property originating from the doped Co atoms.[16]
Recently, catalytic applications of the metal-doped ZIF materials
were also reported; the Cu-doped ZIF-8 (Cu/ZIF-8) exhibited
catalytic activity for condensation reactions. The Cu2+ ions
positioning randomly in ZIF functioned as Lewis acids in the
Friedländer reaction to promote condensation of 2-
aminobenzophenone and methylene.[17] Partial replacement of
Zn with Fe ions in ZIF-8 was also applied for the catalysis of
oxygen reduction in acidic solutions where imidazole-bonded Fe
atoms, in the form of FeN4, are likely to work as active sites for
oxygen-reduction catalysis.[18] These reports imply that the
metal-doped ZIF-8 can exhibit catalytic activity from the defects
of the metal-imidazole complexes.
On the other hand, ZIF has been examined for drug
delivery and bioimaging, exploiting its porous structure and
structural sensitivity to the environmental changes. Delivery of
anti-cancer drugs was performed using ZIF-8 as a porous host
matrix in which drugs were highly loaded and then were
released progressively by pH triggering.[19,20] Its sensitivity to
cytoplasmic stimuli makes ZIF promising for a drug carrier
accessible to the target cell. In a similar fashion, cell imaging
was achieved by releasing fluorescent dye in response to the
metabolic products. Mao et al. reported imaging of the dynamics
of the mitochondrial ATP in live cells with the use of a ZIF-90
enclosing rhodamine B. The mitochondrial ATP has higher
coordination affinity than imidazole-2-carboxyaldehyde, a linker
of ZIF-90, to collapse the ZIF-90 framework, resulting in the
release of fluorescent dye.[21] Release of the fluorescent dye
could also be achieved by a pH stimulus when ZIF-8 was
adopted as a cargo of the fluorescent molecules.[22] The
outcomes of these biomedical applications along with the low
toxicity and stability in neutral solution would open the possibility
of further use of ZIF-8 for other biological applications.
Introduction
The zeolitic imidazolate framework (ZIF), a subclass of
metal organic frameworks (MOFs), possesses zeolite topologies
and general characteristics of MOFs, such as chemical and
thermal stability, large surface area, unimodal porosity, and
controllable chemical functionality.[1,2] Many ZIF materials have
been explored because of their high potential in various
applications as porous materials.[3–7] ZIF-8 is a subtype of ZIF
materials whose ordered, porous structure was constructed
through the assembly of Zn ions with 2-methylimidazole linkers.
Recently, ZIF-8 has attracted research interest because of its
ease of synthesis. Specifically, a water-based synthesis method
reported by Lai et al. offers a facile way to prepare ZIF-8 while
allowing designer modification.[8] According to this method, ZIF-8
nanocrystals on the order of tens of nanometres can be
produced by mixing the Zn ion source and 2-methylimidazolate
together at room temperature for several minutes. This water-
based method surmounts the disadvantages of other methods,
including
the
solvothermal,[9]
microwave,[10,11]
and
[a]
[b]
Changjoon Keum and Prof. Sang-Yup Lee
Department of Chemical and Biomolecular Engineering, Yonsei
University
50 Yonsei-ro, Seodaemun-gu, Seoul, Korea 03722
E-mail: leessy@yonsei.ac.kr
Sangwoo Park
Korea Basic Science Institute, Gwangju Center
77 Yongbong-ro, Buk-gu, Gwangju, Korea 61186
† Authors are equally contributed.
In this study, the Cu-doped ZIF-8 (Cu/ZIF-8) was
demonstrated as a peroxidase-like catalyst exhibiting oxidative
catalytic activity and was exploited for cancer-cell imaging
through the interaction with reactive oxidative species (ROS) in
Supporting information for this article is given via a link at the end of
the document.
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the cell. Previously, biological cofactors were needed to display
catalytic oxidative activity such that the well-known biological
cofactor of hemin (or heme) has been involved in the MOF
(including ZIF-8) through methods of cofactor solution wetting[23]
or solvothermal synthesis[24–26]. These approaches utilized MOF
materials as a simple support to which the heterogeneous
species were implemented physically; thus, these hybrid MOFs
had heterogeneous active sites and the chemical characteristics
of ZIF materials were not extensively exploited for the
expression of catalytic activity.[27] We report here that Cu/ZIF-8
exhibits catalytic oxidative activity by itself without other
biological cofactors and that the catalytic activity of Cu/ZIF-8 with
low toxicity can be exploited for the imaging of cancer cells. The
catalytic activity of Cu/ZIF-8 was characterized and
photoluminescence generated by the oxidation reaction in the
target cell was visualized.
Figure 2. N2 adsorption isotherm of Cu/ZIF-8 and ZIF-8 at 77 K.
indicative of Cu doping to ZIF-8.[17] The SEM images of Cu/ZIF-8
show that the synthesized nanocrystals have rhombic
Results and Discussion
a
dodecahedron structure, which is a typical morphology of ZIF-8
(Figure 1a).[28] The synthesized Cu/ZIF-8 has a diameter of 160
± 47 nm which is slightly larger than that of ZIF-8 (151 nm). This
slight increase of Cu/ZIF-8 is due to the retarded nucleation
when Cu was added in the preparation of Cu/ZIF-8 crystals.[17]
Except for a slight increase in size, no other morphology
changes were observed after Cu doping compared to the intact
ZIF-8. The powder X-ray diffraction (XRD) patterns of ZIF-8 and
Cu/ZIF-8 also match each other, indicating little change in the
crystal structure after Cu doping (Figure 1b). The invariance of
the morphology and crystal structure indicates that doping with
Cu ions does not significantly change the ZIF-8 crystalline
structure. Doping of the Cu ions in Cu/ZIF-8 is confirmed from
atomic mapping (Figure 1c).
Characterization of Cu/ZIF-8
The Cu/ZIF-8 nanocrystals were prepared by mixing metal
sources (Zn and Cu content: 90 and 10%, respectively) to the
Hmim solution. The mixture solution became cloudy and finally
formed a turbid suspension of Cu/ZIF-8 or ZIF-8 precipitates
after 1 h of mixing. The obtained ZIF-8 and Cu/ZIF-8 were
coloured white and beige, respectively. The colour change is
The specific surface area and pore size of the Cu/ZIF-8
were
evaluated
from
BET
experiment.
The
N2
adsorption/desorption curves at 77 K showed a reversible type-I
isotherm (Figure 2).[5] This reversible type-I isotherm indicates
that the prepared ZIFs have relatively small surface area and the
N2 adsorption is mostly determined by the accessible micropore
volume, which is a general character of zeolites and MOFs.[28] It
is likely that N2 adsorbs on micropores at low pressure and on
intrapores at high pressure. From the isotherm, the pore volume
and pore size of ZIF-8 and Cu/ZIF-8 were determined, and are
summarized in Table 1. The pore volume increased after Cu
doping, presumably due to the generation of crystal defects and
Table 1. Porosity of Cu-doped/undoped ZIF-8
ZIF-8
Cu/ZIF-8]
Pore volume (cc/g)
Pore size (Å )
6.07 × 10-1
6.38
6.85 × 10-1
6.28
Figure 1. Shape and crystalline structure of Cu/ZIF-8. (a) SEM image of
Cu/ZIF-8 (inset: magnified image of Cu/ZIF-8 showing dodecahedron
structure), (b) X-ray diffraction spectra of ZIF-8 and Cu/ZIF-8, (c) Copper
mapping on a Cu/ZIF-8 nanoparticle.
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the subsequent destruction of micropores by replacement of Zn
with Cu.[29] The crystal defects may be caused by the difference
in the coordination geometry of these two metal ions; Zn is
tetrahedral and Cu is trigonal-pyramidal.[30] On the other hand,
the pore size changed little (Δd = 1.6%), suggesting that the
main frameworks of ZIF-8 were not considerably deformed.
The amount of the doped Cu in ZIF-8 was measured by
inductively coupled plasma mass spectrometry (ICP-MS). The
content of Cu in Cu/ZIF-8 was 5.1 wt%, which corresponds to
the presence of 221 × 10-9 mol of Cu per 1 mg of Cu/ZIF-8. This
small number explains the little change in XRD peaks after Cu
doping; most of Cu/ZIF-8 keeps ZIF-8 framework even after
doping of Cu.
Catalytic activity of Cu/ZIF-8
Figure 3. Profiles of the DAP produced by the catalytic function of Cu/ZIF-8.
Doping of Cu to ZIF-8 partially replaces Zn ions to
generate Cu-Nx complexes. This Cu–Nx generates vacancies[31]
that may work as active sites for the oxidative catalysis. The
oxidative catalytic activity from Cu/ZIF-8 was verified through a
series of control experiments in which undoped ZIF-8 and other
components for the catalysis were applied. For the catalytic
activity test, 0.1 mg of Cu/ZIF-8 or ZIF-8 was dispersed in the
1.7 mL of OPD solution (10 mM) in HEPES buffer (50 mM, pH
7.2). Catalytic oxidation of OPD was initiated by the addition of
0.2 mL of H2O2, whose concentration became 2 mM in the
reaction mixture. The linear increase in 2,3-diaminophenazine
(DAP), the oxidized product of OPD, concentration was
observed only when Cu/ZIF-8 was added to the substrate
mixture of OPD and H2O2 (Figure 3). In contrast, little production
of DAP was observable when ZIF-8 was applied. This supports
the idea that the doped Cu ions work as catalytic active sites
while the Zn framework has little activity in itself. Catalytic
oxidation in the presence of H2O2 proposes generation of the
active Cu-peroxo intermediate species and subsequent
oxidation occurs, like other catalysts containing copper
center.[32,33] It is already known that the decomposition of H2O2
and the subsequent production of active metal-peroxo species
Figure 4. Apparent reaction rate profiles and double reciprocal plots of OPD oxidation by Cu/ZIF-8. The reaction kinetic assay
was conducted based on the Michaelis-Menten kinetic model. The apparent reaction rate (v0) and [E0]/v0 were plotted (a, c)
for variable OPD concentrations at fixed concentrations of H2O2 (1, 2, and 5 mM), and (b, d) for variable H2O2 concentrations
at fixed concentrations of OPD (85, 425, and 850 μM), respectively.
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occur only when the metal atom is not fully coordinated with
ligands.[31] Therefore, the imperfect coordination of Cu atoms to
imidazoles in Cu/ZIF-8 nanocrystal presumably affords a free
site for the coordination with H2O2, which leads to the expression
of peroxidase-like activity.
푘푐푎푡 [퐶푎푡푎][푂푃퐷][퐻2푂2]
푣0
=
(1)
퐻
푂
2
2
퐾푀푂푃퐷 퐻2푂2 + 퐾푀
푂푃퐷 + 푂푃퐷 퐻2푂2
[
]
[
]
[
][ ]
The determined values of kinetic parameters, kcat and KM,
The catalytic reaction mechanism was investigated from
the plots of the apparent steady-state reaction rates that were
measured with the variation in OPD and H2O2 substrate
concentrations. The reaction rates (v0) were determined from the
slope of the linear DAP profiles at the early stage of reaction,
wherein one substrate concentration varied while the other was
fixed. The bi-substrate Michaelis–Menten kinetics model was
applied to describe the reaction kinetics, like other peroxidase-
like catalysts. The reaction rates of the oxidative catalysis fit well
to the Michaelis–Menten kinetics model shown in Figure 4(a)
and (b); the reaction rate linearly increased at low concentration
and reached the maximum with the increase in the OPD and
H2O2 concentrations. This Michaelis–Menten kinetics model
indicates that the reaction rate is substrate-dependent and
implies the generation of reactive intermediate species during
the bi-substrate reaction.
Double-reciprocal plots were drawn to determine the bi-
substrate kinetic parameters of the apparent turnover number
(kcat) and Michaelis constant (KM). The parallel double-reciprocal
plots were sketched with variation in both substrate
concentrations (Figure 4c, d). The parallel double-reciprocal
plots indicate that the Cu/ZIF-8 catalyst obeys the ping-pong
mechanism that conveys the generation of catalytic
intermediates by the association of the substrate to the catalytic
active site. This ping-pong mechanism has generally been
observed in other inorganic and organic peroxidase-mimetic
catalysts wherein H2O2 works as the ROS to produce oxygen
radicals.[28,34] The reaction rate (v0) following the ping-pong
mechanism can be presented by equation (1), and values of KM
and kcat were calculated from the slope and y-intercepts. When
calculating these kinetic parameters, the catalyst concentration
[Cata] was set as the Cu concentration in Cu/ZIF-8 that was
determined by ICP-MS mentioned above.
are summarized in Table 2. Both kcat and KM increased with
substrate concentration such that the catalytic efficiency (kcat/KM)
did not change significantly with variation in the substrate
concentrations. It is notable that KM is much lower than that of
other artificial peroxidase-like catalysts and comparable to that
of natural HRP (see Table 3). Thus, Cu/ZIF-8 is inferred to have
good affinity to the substrates even though it does not have
other attracting/binding motifs for the substrates.
Table 3. kcat and KM values of peroxidase-like catalysts.
catalyst
HRP
substrate
kcat (s-1)
KM (mM)
Ref.
OPD
H2O2
OPD
H2O2
OPD
H2O2
OPD
H2O2
OPD
H2O2
1.70 × 10-3
2.66 × 10-3
1.14 × 10-2
0.88 × 10-2
290.4[a]
377.6[a]
0.39
0.31
1.40
0.47
242
35
35
Cu2O NWMCs
NDAus
35
35
48.7
208.7
41.34
7.13
0.16
0.37
36
36
Mn2+-His bola
Cu/ZIF-8
37
0.11
37
0.13
this work
this work
0.06
[a] mM s-1 mg -2
The catalytic property of Cu/ZIF-8 for OPD oxidation was
further investigated in terms of the activation energy. The
reaction rate (v0) at the early stage of reaction was measured at
various temperatures in the range of 30–50 °C. The reaction rate
was assumed to be a pseudo-first-order reaction at the early
stage of reaction, when the OPD concentration was excessive
compared to the H2O2 concentration. The Arrhenius plot showing
the linear correlation between the logarithm of the reaction rate
constant (k) and inverse temperature (1/T) was drawn, and then
the activation energy of 59.2 kJ·mol-1 was determined from the
slope (Figure 5). This activation energy is comparable to that of
other peroxidase-mimetic catalysts[35,36], but is still higher than
that of graphene-based catalysts (20–28 kJ·mol-1)[37,38] and a
natural peroxidase (27.1 kJ·mol-1).[39] This relatively high
activation energy is presumably due to the oxidation reaction of
the active intermediate represented by the low turnover number
rather than the binding affinity of the substrate to Cu/ZIF-8,
considering the low values of KM. The catalytic oxidation of OPD
is influenced by the microenvironment around the active site
wherein other oxygenated functional groups assist OPD
oxidation.[40,41] Thus, absence of such oxygenated functional
Table 2. Apparent kinetic parameters of Cu/ZIF-8 for catalytic OPD oxidation.
H2O2
kcat (s-1)
KMOPD (mM)
kcat/KMOPD (M-1s-1)
concentration
1 mM
2 mM
5 mM
0.128
0.225
0.299
0.158
0.278
0.353
810.6
809.5
845.8
OPD
kcat (s-1)
KMH2O2 (mM)
kcat/KMH2O2 (M-1s-1)
concentration
85 μM
425 μM
850 μM
0.062
0.180
0.276
0.365
0.845
1.412
170.9
212.8
195.7
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Figure 6. HeLa cell viability assay. HeLa cells were cultured in 96 well plate
containing variable OPD or Cu/ZIF-8 concentrations for 12 h at 37°C. After
incubation, MTT assay was performed and fluorescence intensity at 540 nm
was read. Cells without addition of OPD or Cu/ZIF-8 were used as control.
Figure 5. Arrhenius plot of Cu/ZIF-8 for the OPD oxidation in the temperature
range of 30–50°C. Reaction rate k was determined from the slope of the DAP
production profiles at the early stage of the catalysis. The reaction was
assumed to be a first-order reaction.
was liberated from Cu/ZIF-8, respectively (Supporting
Information, Table S1). From the Cu release property, a dosage
of 0.22 nM of copper ion per single HeLa cell was approximated.
This amount of released Cu species is not critical to cause
substantial cytotoxicity. The HeLa cell viability was higher than
90% at up to 5 μM of Cu ions, which was confirmed from a
supplementary test (SI, Figure S1). This is in agreement with the
reported critical concentration of 70 μM (=4.5 ppm) at which the
cell viability is lower than 90%. [45]
Based on the results of cell viability test, HeLa cell imaging
was conducted using Cu/ZIF-8 and OPD at concentrations of 30
μg/mL and 0.42 mM, respectively. Figure 7(a) shows the optical
and confocal microscopy images of the cells. In the presence of
both Cu/ZIF-8 and OPD, strong emission of the green
photoluminescence was observable after 4 hours. The merged
image confirms that the green fluorescence comes from the cell,
not from the medium. In contrast, very weak green fluorescence
was observable when OPD was solely applied without Cu/ZIF-8,
and no fluorescence was emitted in the absence of OPD (data
not shown). The fluorescence intensity was compared for the
quantitative analysis of the effect of the Cu/ZIF-8 catalyst (Figure
7b). Around 2.5 times stronger fluorescence was observable
when Cu/ZIF-8 was applied. This confocal microscopy analysis
supports that OPD oxidation is promoted by Cu/ZIF-8 in the cell
where the ROS are present.
group around the active Cu site of Cu/ZIF-8 is a potential factor
for the high activation energy and low turnover number.
Cancer cell imaging by Cu/ZIF-8
Cancer cell imaging was demonstrated exploiting the
catalytic activity of Cu/ZIF-8 with the ROS in a cell. Various ROS,
like peroxide and hydroxyl radical (•OH), are generated as
byproducts during the metabolism of oxygen in a cell[42] and can
be utilized for the oxidative reaction of OPD or other organic dye
in the presence of Cu/ZIF-8 catalyst. DAP, the oxidized form of
OPD, emits green photoluminescence that can be used for cell
imaging. Prior to conducting the cell imaging, the toxicity of
Cu/ZIF-8 and OPD was examined using a HeLa cell line. Briefly,
Cu/ZIF-8, OPD, and
a mixture of these two at various
concentrations were added to the HeLa cell suspension (1x
corresponds to OPD = 0.14 mM and Cu/ZIF-8 = 10 μg/mL) in a
96-well plate and then incubated 12 h at 37 °C. After incubation,
MTT assay was conducted to measure the cell viability. The
HeLa cells showed good cell viability (average 92.7%) under
exposure to 0.5x–5x of OPD (Figure 6). Because OPD is an
aromatic diamine with low toxicity of median lethal dose (LD50) of
44 mg/mL,[43] the high cell viability is reliable. In contrast, Cu/ZIF-
8 showed considerable toxicity over 50 μg/mL (5x) concentration,
although other studies reported low cytotoxicity of ZIF-8.[44] This
low cell viability might come from the catalysed oxidation of
cellular component by ROS in the presence of Cu/ZIF-8. Without
the Cu/ZIF-8 catalyst, such significant oxidation would not occur;
however, the presence of the catalyst promotes oxidation of
cytoplasmic cellular components, resulting in a decrease in cell
viability. The dissociated Cu species from the Cu/ZIF-8 also
need to be counted, which may change the viability of the HeLa
cell. We conducted ICP of the supernatant of the Cu/ZIF-8
suspension after 12 h incubation in pH 5.0 and 7.4 phosphate
buffer solutions and found that 26.3 wt% and 18.4 wt% of Cu
As a control, the HEK293T cell line was tested for imaging
through the same protocol. Optical and fluorescence microscopy
images in Figure 7(c) show that little fluorescence was emitted
from the HEK293T cells. This is probably because fewer ROS
are present in the cytoplasm of the HEK293T cells, or less
endocytosis of the OPD and Cu/ZIF-8 occurred. Although the
clear reason for the weak fluorescence from HEK293T cell could
not be rationalized, the control experiment supports that Cu/ZIF-
8 can be exploited for cancer cell imaging where ROS are
abundant to oxidize OPD.
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Figure 8. Photoluminescence emitted from the DAP (oxidized OPD) by
various ROS in the presence/absence of Cu/ZIF-8 nanocrystals. Inone
represents the intensity of the photoluminescence in the absence of Cu/ZIF-8
nanocrystals.
common ROS present in a cancer cell.[42] These ROS were
prepared chemically or biologically (SI, methods for preparation
of ROS) and subsequently reacted with OPD in the presence of
Cu/ZIF-8. The photoluminescence (λem = 550 nm) intensity from
DAP was monitored to identify the oxidation of OPD by the
active ROS species. All ROS oxidized OPD, even in the
absence of Cu/ZIF-8. However, remarkable oxidation of OPD
showing outstanding photoluminescence occurred when H2O2
was applied in the presence of Cu/ZIF-8. By contrast, enhanced
oxidation of OPD was hardly observable for the other ROS even
in the presence of Cu/ZIF-8 (Figure 8). This result strongly
supports that H2O2 is the key ROS in the HeLa cell that
produces oxidative active species through the interaction with
Cu/ZIF-8. The sensitivity of Cu/ZIF-8 nanocatalyst to H2O2 is
comparable to other probes for fluorescence imaging of
intracellular H2O2 such as hemin-decorated gold nanoparticle[46]
and mesoporous silica nanoparticles decorated with ferrocene[47]
or a cyanine-derivative.[48]
Conclusions
In summary, reaction kinetics and cancer cell imaging data
demonstrated that the Cu/ZIF-8 nanocrystals could be used as
an oxidative catalyst that has low toxicity. The Cu atoms doped
in ZIF-8 working as active sites to oxidize an organic substance
through selective interaction with H2O2, along with the stable
ZIF-8 structure in the aqueous environment, were responsible
for the high catalytic activity and reactivity with the ROS rich in a
cancer cell. In comparison to other MOF-based oxidative
catalysts requiring the heme cofactor for expression of the
oxidative activity, this Cu/ZIF-8 could be prepared without other
biological substances, which enabled facile preparation of the
catalysts without complication. Furthermore, the considerable
affinity to the substrates, including specificity to H2O2 among
various ROS in a cell, could confirm the capability of Cu/ZIF-8
Figure 7. HeLa cell imaging using Cu/ZIF-8 and OPD. (a) Confocal
microscopy images of the HeLa cells in the presence of both Cu/ZIF-8 and
OPD (top) and OPD only (bottom) (scale bar = 25 μm). The images are optical,
fluorescence, and merged microscopy images, from left to right. (b)
Fluorescence intensity from the HeLa cells in the presence of Cu/ZIF-8 and/or
OPD. Fluorescence intensity was measured from 100 randomly selected cells
for each sample. (c) Confocal microscopy images of the HEK293T cells in the
presence of both Cu/ZIF-8 and OPD (top) and OPD only (bottom) (scale bar =
25 μm).
To identify the ROS causing the OPD oxidation in a cell,
-
tests were conducted using H2O2, •OH, 1O2, and O2 , which are
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Catalytic activity measurement was performed using 0.1 mL of Cu/ZIF-8
suspension (1 mg/mL) in a HEPES buffer (50 mM, pH 7.2) and 1.7 mL of
OPD solution (10 mM) in the same HEPES buffer at various
concentrations. The oxidation was initiated by dropping 0.2 mL of H2O2
solution to the mixture of Cu/ZIF-8 and OPD solutions. The reaction rate
was determined from the slope of the OPD oxidation at the initial stage.
The kinetics of the catalytic reaction were analysed based on the
Michaelis–Menten equation while measuring the reaction rate with
respect to the variation in the OPD and H2O2 concentrations. The kinetic
parameters of the Michaelis–Menten equation were determined from the
double-reciprocal plot.
for imaging of HeLa cell. The catalytic activity and low toxicity of
this easy-to-prepare Cu/ZIF-8 make this nanocrystal an
appealing heterogeneous catalyst for microbiological analysis as
well as conventional oxidative catalysis used in various chemical
processes.
Experimental Section
Chemicals
Cell viability test and cell imaging
The reagents for Cu/ZIF-8 synthesis were purchased from Sigma-Aldrich:
zinc nitrate hexahydrate (Zn(NO3)2·6H2O, reagent grade, 98%), copper
nitrate trihydrate (Cu(NO3)2·3H2O, puriss. p.a., 99-104%) and 2-
methylimidazole (Hmim, 99%). In the catalysis experiments, o-
phenylenediamin (OPD, 98%, Sigma) was used as a substrate. All
chemicals were used as received.
The human embryonic kidney cells (HEK-293T cells) and human cervical
cancer cells (HeLa cells) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM)/F12 (1:1) supplemented with 10% fetal bovine serum
(FBS), 1% penicillin, and streptomycin under 5% CO2 at 37 °C. For the
cell
viability
test,
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was conducted. Briefly, the HeLa
cells were seeded on a 96-well plate (1 × 104 cells per well) in 100 μL of
DMEM cell culture medium. After 24 h, the medium was changed with a
fresh DMEM medium mixture containing the proper concentrations of
OPD, Cu/ZIF, and H2O2, and then the cells were incubated for another 12
h. After that, the medium was washed with the DPBS buffer solution and
was replaced with DMEM solution (80 μL) containing MTT solution (0.5
mg/mL) for another 4 h. The medium was removed, leaving 25 μL, and
then 50 μL of DMSO was added to the remaining medium in order to
dissolve the formazan. A sunrise microplate reader (Tecan, Austria) was
used to record the absorbance intensity at 540 nm. The experiments
were repeated at least three times. The cell viability (%) was determined
as % viability [= (OD of treated cell - OD of blank)/(OD of untreated cell -
OD of blank) × 100], where OD is the optical density.
Preparation of Cu/ZIF-8
To prepare Cu-doped ZIF-8, 35 mL of 2 M Hmim solution and 10 mL of
metal precursor solution containing 180 mM Zn(NO3)2 and 20 mM
Cu(NO3)2 were prepared. The metal precursor solution was mixed with
the Hmim solution under vigorous stirring and aged 1 h. The final molar
ratio of the synthesis solution was Zn:Cu:Hmim:water = 0.9:0.1:35:1280.
After 1 h of stirring, the Cu/ZIF-8 crystals were precipitated and
separated by centrifugation (9,000 rpm, 15 min). The remaining
chemicals were washed off with deionized water three times. After
vacuum drying for 12 h at 60 °C, beige coloured powder was obtained.
Undoped ZIF-8 was synthesized through the same process without
addition of Cu(NO3)2.
Characterization
For the imaging studies of HeLa and HEK-293T cells, the cells (1 × 105
cells per dish) were seeded on the cover glass in a 12-well plate and
cultured for 1 day. The medium was then replaced with fresh DMEM
containing OPD and/or Cu/ZIF-8 (0.42 mM and 30 μg/mL, respectively).
All time-dependent experiments were performed by washing cells with
DPBS before the observation. Confocal images were obtained using a
laser-scanning confocal microscope system (Leica TCS SP5
AOBS/Tandem, Leica Microsystems, Germany) at Korea Basic Science
Institute, Gwangju Center.
The crystalline structure of the as-prepared Cu/ZIF-8 was characterized
by an X-ray diffraction analyser (XRD, Ultima IV, Rigaku) equipped with a
CuKα radiation source (λ = 1.542 Å). XRD spectra of Cu/ZIF-8 and ZIF-8
were obtained over the ranges of 5° to 55° (step size: 0.02°, rate: 2°/min).
For the characterization of surface area and pore volume, Brunauer–
Emmett–Teller (BET) analyses on ZIF-8 and Cu/ZIF-8 were conducted.
The dried powder samples were degassed at 150 °C in a vacuum for 3 h,
and then N2 adsorption/desorption isotherms were obtained using a
surface-area and pore-size analyser (BET, Autosorb-iQ 2ST/MP,
Quantachrome). The isotherms were analysed using the BET method
and the pore volume was determined by the Horvath–Kawazoe equation.
The microscopic image analysis and Cu ion characterization of Cu/ZIF-8
were performed by transmission electron microscopy (TEM, JEM-ARM
200F, JEOL, 200kV) equipped with EDX (Inca, Oxford, U.K.) and
scanning electron microscopy (SEM, JSM-6701F, JEOL). To prepare the
sample for TEM analysis, Cu/ZIF-8 powder was re-dispersed in H2O and
then dropped on the nickel grid. The nickel grid was used to identify the
Cu in the Cu/ZIF-8. The content of Cu ions was examined from
inductively coupled plasma mass spectrometry (ICP-MS, ICP-MS 7900,
Agilent).
Toxicity test of copper released from Cu/ZIF-8
HeLa cell viability against the copper ion liberated from Cu/ZIF-8 was
conducted with exposure of the cells to copper nitrate trihydrate solutions.
In the same method as described above, HeLa cells were cultured in a
DMEM medium mixture containing various concentrations of copper
nitrate trihydrate for 12 h, and then the MTT assay was performed. To
evaluate the quantity of released Cu ions from Cu/ZIF-8, ICP-MS
analyses on the liberated copper ion were conducted. First, the content
of Cu and Zn atoms in Cu/ZIF-8 nanocrystals were determined. Cu/ZIF-8
nanocrystals were dissolved in the mixture of concentrated HNO3 and
H2O2 fully to a concentration of 1 mg/mL, and then the dissolved solution
was analysed using inductively coupled plasma mass spectrometry (ICP-
MS). To evaluate the amount of Cu and Zn liberated from the Cu/ZIF-8 in
cytosol, 4 mg of Cu/ZIF-8 nanocrystals were incubated in 4 mL of pH-
controlled phosphate buffer saline solutions at pH 7.4 and 5.0,
respectively. After 12 h of incubation at 37 °C, the supernatants of the
Cu/ZIF-8 solution were collected and the concentrations of the liberated
metal ions were measured.
Catalytic oxidative activity of Cu/ZIF-8
The catalytic oxidative activity of Cu/ZIF-8 was evaluated from the
oxidation of o-phenylenediamine (OPD), which is oxidized to 2,3-
diaminophenazine (DAP), displaying a yellow colour. The colour change
following the OPD oxidation was monitored using
spectrophotometer (S-3100, Scinco, λabs = 420 nm) under mild stirring.
a
UV−vis
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10.1002/asia.201800749
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Entry for the Table of Contents (Please choose one layout)
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Copper-doped zeolitic imidazolate
framework-8 (Cu/ZIF-8) nanocrystal
exhibiting oxidative catalytic activity
was used as imaging agent for
reactive oxygen species of cancer
cell.
Changjoon Keum, Sangwoo Park, Sang-
Yup Lee*
Page No. – Page No.
Cancer-Cell Imaging Using Copper-
Doped Zeolite Imidazole Framework-8
Nanocrystals Exhibiting Oxidative
Catalytic Activity
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